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PLC-β signaling is generally thought to be mediated by allosteric activation by G proteins and Ca2+. While availability of the PIP2 substrate is limiting in some cases, its production has not been shown to be independently regulated as a signaling mechanism. WNK1 protein kinase is known to regulate ion homeostasis and cause hypertension when expression is increased by gene mutations. However, its signaling functions remain largely elusive.
Using diacylglycerol-stimulated TRPC6 and inositol trisphosphate-mediated Ca2+ transients as cellular biosensors, we show that WNK1 stimulates PLC-β signaling in cells by promoting the synthesis of PIP2 via stimulation of phosphatidylinositol 4-kinase IIIα. WNK1 kinase activity is not required. Stimulation of PLC-β by WNK1 and by Gαq are synergistic; WNK1 activity is essential for regulation of PLC-β signaling by Gq-coupled receptors and basal input from Gq is necessary for WNK1 signaling via PLC-β. WNK1 further amplifies PLC-β signaling when it is phosphorylated by Akt kinase in response to insulin-like growth factor.
WNK1 is a novel regulator of PLC-β that acts by controlling substrate availability. WNK1 thereby coordinates signaling between G protein and Akt kinase pathways. Because PIP2 is itself a signaling molecule, regulation of PIP2 synthesis by WNK1 also allows the cell to initiate PLC signaling while independently controlling the effects of PIP2 on other targets. These findings describe a new signaling pathway for Akt-activating growth factors, a mechanism for G protein-growth factor crosstalk and a means to independently control PLC signaling and PIP2 availability.
Mammalian WNK kinases are a family of four serine-threonine protein kinases with an atypical kinase active site [1–3]. Mutations in WNK1 and WNK4 cause the autosomal-dominant disease pseudohypoaldosteronism type 2 (PHA2), which is characterized by hypertension and hyperkalemia . WNKs regulate various ion transporters through both kinase activity-dependent and -independent mechanisms [3, 4]. For example, WNK1 and 4 phosphorylate and activate Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1), which in turn activate cation-chloride co-transporters NCC and NKCC [5–7]. Through kinase-independent events, WNK1 binds and somehow activates SGK kinase, causing it to activate the epithelial Na+ channel ENaC ; and WNK1 and 4 interact with the endocytic scaffold protein intersectin to enhance endocytosis of the renal K+ channel ROMK . Dysregulation of ion transport in the kidney contributes to the hypertension and hyperkalemia of PHA2.
The functions of WNKs extend beyond regulation of ion homeostasis. WNK1 is ubiquitously expressed, and homozygous gene inactivation in mice causes cardiovascular developmental arrest and embryonic death . WNK1 activates ERK5, and knockdown of WNK1 decreases activation of ERK5 by epidermal growth factor receptors . WNK2 binds the small GTPases RhoA and Rac1. WNK2 knockdown decreases RhoA activation, but promotes Rac1 activation . The Gq-coupled angiotensin II receptor regulates the activity of the NCC co-transporter through the WNK4 signaling pathway , indicating functional interactions between WNK and G protein signaling pathways.
Upstream regulation of the WNKs is incompletely understood, and it is likely that several distinct mechanisms of WNK activation are used in different cells [3, 4]. WNK1 is a substrate of Akt kinase [14, 15], which is a target of phosphatidylinositol 3-kinase (PI3K)-activating growth factors such as insulin and insulin-like growth factors (IGFs). Insulin and IGFs activate PI3K, and its PIP3 product recruits and activates Akt to phosphorylate WNK1 at threonine-58. It has been reported that Akt-catalyzed phosphorylation of WNK1 at T58 alters the ability of WNK1 to regulate membrane trafficking of ENaC and ROMK channels [8, 16].
Phospholipase C-βs (PLC-βs) are important effectors of G protein-coupled receptor (GPCR)-mediated signaling pathways [17, 18]. They hydrolyze PIP2, itself a signaling molecule, to form two other second messengers, diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). PLC-βs are stimulated by Gα subunits of the Gq family, by Gβγ subunits of the Gi’s and, in the case of PLC-β2, by Rac [17–19]. The concentration of PIP2 in the plasma membrane is limiting for PLC-β activity, and re-synthesis of the substrate initiated by phosphatidylinositol-4-kinase (PI4K) after depletion is important for sustained PLC-β signaling [20, 21]. This dependence on substrate concentration agrees with in vitro studies showing that PLC activity increases linearly with the concentration of PIP2 regardless of stimulation by G proteins or Ca2+ [22, 23]. However, it is not known whether PIP2 production can be independently regulated as a signaling mechanism and, if so, by what physiological stimulus.
We show here that WNK1 stimulates PLC-β signaling by promoting the synthesis of PIP2 via stimulation of PI4KIIIα. Stimulation of PLC-β by WNK1 and by Gαq-GTP is thus synergistic, and WNK1 activity is essential for regulation of PLC-β signaling by Gq-coupled receptors. Phosphorylation of WNK1 by the PI3K-Akt kinase cascade enhances the ability of WNK1 to synergistically regulate PLC-β signaling via synthesis of PIP2. Regulated control of substrate availability represents a novel mechanism for regulation of PLC-β activity and for crosstalk between Gq-coupled receptors and receptors that activate the Akt kinase pathway.
The TRPC6 channel is directly activated by DAG, a product of the PLC-catalyzed hydrolysis of PIP2 . Inspired by a serendipitous observation that WNK1 knockdown altered Gq signaling, we used TRPC6 as a biosensor to examine the role of WNK1 in PLC-β signaling in native cell membranes. Full-length WNK1 is difficult to purify, and we tested the effect of WNK1 on TRPC6 by intracellular application of a purified fragment of rat WNK1 consisting of residues 1–491 (WNK11–491), which contains the Akt-phosphorylation site, the protein kinase domain and a N-terminal proline-rich domain (Figure 1A). As shown for HEK293 cells in Figure 1B, intracellular WNK11–491 increased TRPC6 current density, peaking in these cells at around 1–2 min and lasting for > 5 min. The half-maximal concentration of WNK11–491 for activation of TRPC6 was ~50 nM (Figure 1C). WNK11–491 also activates TRPC6 in membranes of HeLa cells (see Figure 6) and mouse embryo fibroblasts (see Figures 2C and 2D). TRPC6 was also activated by GTPγS, consistent with Gq-PLC-β signaling to produce DAG, and by the muscarinic agonist carbachol in cells that express the Gq-coupled M3 muscarinic cholinergic receptor (Figure 1B).
Application of WNK11–491 to the cytoplasmic face of detached plasma membranes also activated TRPC6 single channel activity in membranes of cells that express TRPC6 (Figure S1A) but not in membranes from control cells. Thus, the cellular components required for activation of TRPC6 by WNK1 are contained in or are bound to the excised plasma membrane patch. Stimulation of TRPC6 was selectively blocked by antibody against amino acid residues 160–176 of WNK1 (Figure S1B). Purified WNK11–491 carrying a kinase-dead K233M mutation [1, 9] retained full ability to activate TRPC6 (Figure S1C), indicating that kinase activity of WNK1 is dispensable.
Several experimental results argue that the activation of TRPC6 by WNK1 is mediated by PLC-β-catalyzed production of the known TRPC6 activator DAG. First, activation of TRPC6 by WNK11–491 was blocked by the PLC inhibitors U73122 (Figure 1D) and edelfosine  (Figure 1E). Application of 1-oleoyl-2-acetyl glycerol (OAG), a soluble and membrane-permeant DAG, restored TRPC6 activity in the continued presence of U73122, which excludes effects of U73122 on the channel itself or on other components upstream of PLC. Second, because edelfosine acts slowly , we could rescue WNK1 signaling by addition of purified PLC-β1 after endogenous PLC was inhibited (Figure 1E). After edelfosine pretreatment, application of both purified PLC-β1 and WNK11–491 caused robust activation of TRPC6. Application of PLC-β1 without WNK1 had no effect, indicating that both WNK1 and PLC-β1 are required for activation of TRPC6. Third, PLC activity is dependent on cytosolic Ca2+ [17, 18], and removal of Ca2+ blocked TRPC6 stimulation by WNK11–491 but not direct stimulation by OAG (Figure 1F). Based only on the data above, WNK11–491 might activate TRPC6 by protecting DAG from hydrolysis by DAG lipase or phosphorylation by DAG kinase. However WNK11–491 did not alter the potency with which OAG activates TRPC6 (Figure 1G), indicating that its effect is at the level of PLC. Together, these results provide compelling evidence that WNK1 activates TRPC6 by stimulation of PLC to produce DAG, the proximal activator.
We also measured intracellular Ca2+ release triggered by IP3, another product of PIP2 hydrolysis, as a read-out for WNK1’s effects on PLC activity. Microinjection of anti-WNK1 antibody or knocking down endogenous WNK1 by small interfering RNA (siRNA) reduced the intracellular Ca2+ increases in response to carbachol (Figures S1D–F). Endogenous WNK1 is thus required for normal PLC-β signaling in response to stimulation of Gq.
The PLC-βs are activated by Gαq [17, 18]. GDPβS, which blocks G protein activation, inhibited the stimulation of TRPC6 by WNK11–491 (Figure 2A). GTPγS stimulated the TRPC6 current without added WNK1, and stimulation was blocked by anti-WNK1 antibody (Figure 2B, Figures S2A and S2B). These data suggest both that some G protein is required for WNK1 stimulation of TRPC6 and that endogenous WNK1 is required for TRPC6 stimulation by a G protein. To determine whether WNK1 requires activation of PLC-β by Gq/11 in cells, we measured TRPC6 regulation by WNK1 in murine Gαq−/−-Gα11−/− embryonic fibroblasts . In intact Gαq/11-null cells, WNK11–491 failed to activate TRPC6, and expression of wild type Gαq restored activation by WNK1 (Figure 2C). When the constitutively active Q209L-Gαq mutant was expressed, addition of WNK11–491 further activated TRPC6 (Figures S2C and S2D), which argues that WNK1 and Gαq act in parallel rather than in series to stimulate DAG synthesis by PLC-β.
When WNK1 was depleted in the Gαq/11-null cells by siRNA, neither WNK11–491 nor Gαq-GTPγS alone stimulated TRPC6 substantially, only simultaneous addition of both activators produced major TRPC6 opening (Figure 2D). Moreover, the Gq inhibitor RGS4 blocked activation of TRPC6 by WNK1 in cells and the N128G mutant of RGS4  had no effect (Figures S2E and S2F). In detached patches, inhibition by RGS4 was reduced and delayed by expression of the RGS-insensitive G188S mutant of Gαq [28, 29] (Figures 2E and 2F). Thus, WNK1 and activated Gαq stimulate TRPC6 synergistically.
Intracellular application of anti-WNK1 antibody markedly blunted the activation of TRPC6 by agonist through the expressed M3 muscarinic receptor (Figure 3A), the α1A-, α1B-adrenergic receptors and H1 histaminergic receptor in HEK cells (Figures S3A–C) or through endogenous muscarinic receptor in HeLa cells (Figure S3D). Conversely, increased WNK1 activity potentiated activation of TRPC6 by M3 receptor. Intracellular application of 3 nM WNK11–491, which is too low to activate TRPC6 by itself, approximately doubled the TRPC6 current elicited by M3 muscarinic stimulation (Figure 3B). Thus, some level of endogenous WNK1 activity is necessary for full Gq-PLC-β signaling and additional WNK1 potentiates Gq signaling in cells.
Anti-WNK1 antibody did not significantly inhibit activation of TRPC6 by the Gi-coupled M2 muscarinic receptor (Figure S3E). However, Gi is a less effective activator of PLC-βs [17, 18], and TRPC6 activation by the M2 receptor is much smaller than that by the Gq-coupled receptors (Figure S3E vs Figure 3A). This apparent selectivity of anti-WNK1 antibody for Gq-coupled receptors suggests that WNK1 is needed to support the higher flux of the Gq-PLC-β pathway but is not detectably required for Gi-PLC-β signaling.
While these data are consistent with the stimulation of PLC-β activity by WNK1, we did not find any effect of purified WNK11–491 on the activity of purified PLC-β1, 2 or 3; its regulation by Gαq or Gβγ; or regulation of Gq by M1 muscarinic receptors (Figure S4).
The interdependence and mutual potentiation of TRPC6 activation by the Gαq and WNK1 pathways suggests that they each increase the activity of PLC-β by distinct mechanisms. Gαq allosterically stimulates the catalytic activity of PLC-β. We therefore asked whether WNK1 acts by increasing the concentration of the PIP2 substrate. Generation of phosphatidylinositol 4-P (PI4P) by PI4K is believed to be the first step in the synthesis of PIP2 . Pretreatment of cells with the PI4K inhibitor wortmannin inhibited activation of TRPC6 by WNK11–491 (Figure 4A) as did simultaneous application of an anti-PI4P antibody  (Figure 4B). Last, intracellular application of PI4KIIIβ activated TRPC6 even in the absence of added WNK1, and pretreatment with wortmannin prevented activation (Figure 4C). PI4P synthesis is thus involved in the stimulation of TRPC6 by WNK1, and WNK1 can be mimicked by addition of a PI4K.
We used the GFP-tagged PH domain of PLC-δ1 and confocal imaging to examine the role of WNK1 in the degradation and re-synthesis of PIP2 in the plasma membrane. The PLC-δ1-PH domain binds both PIP2 and IP3 with relatively high affinity and specificity [32–34]. In cells that express M3 receptor, addition of carbachol decreased GFP-PLC-δ1-PH fluorescence at the plasma membrane, indicating reduced PIP2, and increased fluorescence in the cytosol, indicating increased IP3 (Figure 5). Both effects peak ~45 sec after carbachol addition and then decline to pre-stimulus levels, reflecting PLC-β-catalyzed hydrolysis of PIP2 and its subsequent re-synthesis.
Microinjection of anti-WNK1 antibody both delayed and decreased the recovery of GFP-PLC-δ1-PH fluorescence at the plasma membrane after treatment with carbachol. Instead of the monophasic return to baseline in <3 min observed in control cells, cells treated with anti-WNK1 antibody displayed biphasic recovery of plasma membrane fluorescence that was only ~50% complete after 15 min. The return of cytosolic fluorescence to baseline was also biphasic, with a greatly prolonged and incomplete second phase. These data indicate that anti-WNK1 antibody substantially inhibits re-synthesis of PIP2 after a burst of PLC activity. Taken together with the effects of the anti-PI4P antibody, they indicate that WNK1 is necessary to maintain cellular PIP2 synthesis.
The PI4KIIIα isozyme is believed to be primarily responsible for re-synthesis of PIP2 in the plasma membrane during receptor stimulation of PLC . To determine whether WNK1 regulates PI4KIIIα, we studied effects of manipulating WNK1 activity in cells that express recombinant PI4KIIIα, where this isoform is the predominant PI4K activity (~95%) in a light membrane fraction, which includes plasma membranes (Figure 6A). Knockdown of WNK1 by siRNA decreased both PI4K activity and the amount of immunoreactive PI4KIIIα in the light membrane fraction by 35–40% (Figures 6B–E) and increased the abundance of PI4KIIIα in cytosol (Figures 6D and 6E). WNK1 knockdown did not alter the amount of PI4KIIIα in the total post-nuclear supernatant (PNS). These data suggest that WNK1 recruits PI4KIIIα from cytosol to the light membrane fraction, presumably the plasma membrane. Recruitment may be indirect, because we did not detect physical association between PI4KIIIα and purified WNK11–491 in pull-down assays.
WNK1 failed to activate TRPC6 in PI4KIIIα-knockdown cells (Figures 6F and 6G) and expression of PI4KIIIα in the knockdown cells rescued activation by WNK1. These results are consistent with the idea that WNK1 increases PIP2 synthesis by recruitment of PI4KIIIα to its site of action, and that the consequently increased availability of PIP2 synergistically enhances Gq-coupled receptor signaling through PLC-β.
The physiological role of WNK1 in the regulation of Gq-PLC-β signaling was further supported by the finding that IGF1, a known upstream activator of WNK1, enhances the effect of WNK1. Pretreatment with IGF1 in serum-starved cells potentiated the activation of TRPC6 by either carbachol (Figures 7A and 7B) or GTPγS (Figures S5A and S5B), although IGF1 did not alter basal TRPC6 current. Lack of effect on the basal current is probably due to a low basal output from Gq-PLC-β in serum-starved cells, and IGF1 activated TRPC6 in these cells when pre-stimulated by GTPγS (Figure S5C). IGF1 also enhanced carbachol-induced intracellular Ca2+ transients (Figure S5D), consistent with a final common site of action at PLC-β. IGF1 did not increase the cell-surface abundance of TRPC6 (Figure S5E).
Several observations argue that IGF1 potentiates GPCR-Gq signaling through a PI3K-Akt-WNK1-PI4KIIIα sequence. First, wortmannin, at a low concentration selective for PI3K (20 nM), blocked the effect of IGF1 on Gq signaling. Knockdown of WNK1 also blocked the ability of IGF1 to potentiate carbachol’s effect (Figure 7C). Expression of wild type WNK1, but not the T58A mutant WNK1, restored the ability of IGF1 to enhance carbachol activation of TRPC6 in WNK1-knockdown cells (Figure 7D), indicating that the synergistic effect of IGF1 and M3 muscarinic stimulation depends on Akt-mediated phosphorylation of WNK1 at T58. Note that both wild type WNK1 and the T58A mutant can support carbachol activation of TRPC6 in WNK1-knockdown cells. Thus, T58-phosphorylation is not required for WNK1 amplification of Gq-PLC-β signaling, but is indispensable for its potentiation by IGF1.
Knockdown of PI4KIIIα also prevented enhancement of carbachol activation of TRPC6 and intracellular Ca2+ transients by IGF1 (Figure 7E, Figure S5F). These results argue that PI3K-dependent activation of Akt by IGF1 leads to phosphorylation of WNK1 and enhances the ability of WNK1 to synergistically regulate PLC-β signaling by promoting PIP2 synthesis.
Our data establish three new and distinct but inter-related signaling mechanisms: the mutual dependence and synergistic interaction between the Gq-PLC-β and Akt-WNK1 signaling pathways; the regulation of PLC-β activity by modulating the availability of its substrate; and the regulation of PI4KIIIα by WNK1 to control the concentration of PIP2-derived second messengers.
As summarized in Figure 7F, blocking WNK1 activity markedly inhibits PLC-β signaling by Gq-coupled receptors and, conversely, inhibiting Gq blocks PLC-β signaling by WNK1. Inhibition could be exerted by several approaches and at several steps in each pathway, both in cells and in detached membrane patches. In each case, replenishment of the inhibited or depleted species, or stimulation of a downstream component, restored normal activities of both WNK1 and Gq-PLC-β. Importantly, WNK1 can elicit PLC signals without added GPCR agonist, and WNK1 synergistically amplifies the stimulation of PLC-β by Gq.
In contrast to G proteins, WNK1 increases PLC-β activity by increasing the supply of the PIP2 substrate. Because the concentration of PIP2 in the plasma membrane is below Km [22, 23], PLC-β displays first-order substrate kinetics and increase in available PIP2 linearly amplifies PLC-β output. Note that WNK1 only modulates Gq signaling via PLC-β. Other outputs, such as stimulation of a rho GEF , are not involved. WNK1 thus alters the spectrum of Gq outputs as well as its amplitude.
WNK1 promotes synthesis of PIP2 by somehow activating PI4KIIIα (Figures 6 and and7F).7F). PI4KIIIα is important for the re-synthesis of PIP2 following hydrolysis by receptor-stimulated PLC , but how the activity of PI4KIIIα is regulated is unknown. The amount of PI4KIIIα is decreased in membranes from WNK1 knockdown cells, suggesting that regulation of PI4KIIIα by WNK1 may involve its redistribution between plasma membrane and intracellular pools. However, WNK1 must do more than just recruit PI4KIIIα to the plasma membrane because it remains necessary for PLC-β signaling in detached patches. We have no evidence that WNK1 either binds or phosphorylates PI4KIIIα, and additional factors may be involved.
Regardless, our results indicate that the activity of PI4KIIIα is regulated and that its regulation by WNK1 is critical for PLC-β signaling by Gq-coupled receptors. The basal activity of WNK1 provides adequate tonic stimulation of PI4KIIIα to provide PIP2 for normal PLC-β signaling. However, additional WNK1 markedly amplifies signaling by Gq-coupled receptor even at a concentration that does not by itself activate TRPC6 (Figure 3B). In addition, PI3K-activating growth factors such as IGF1 activate WNK1 to further potentiate Gq-PLC-β signaling, probably via Akt-mediated phosphorylation of WNK1 at T58 (Figure 7). These results suggest that WNK1 is an important physiological mediator of the Akt kinase signaling pathway and coordinates Akt signaling with the Gq-PLC-β signaling pathway.
This action of WNK1 represents the first known instance of acute control of PIP2 availability as a mechanism of regulation of cellular DAG and IP3 concentrations without concurrent stimulation of the intrinsic activity of PLC. Application of WNK1 alone stimulates the production of DAG measured using the TRPC6 biosensor by an amount comparable to that initiated by GTPγS (Figure 1B). Some low output of PLC-β, evidently dependent on basal input from Gαq, is adequate to support this WNK1 signaling. By promoting PIP2 synthesis, WNK1 can also acutely modulate the amplitude of receptor-stimulated PLC-β signaling and/or its duration. Inhibition of endogenous WNK1 by anti-WNK1 antibody blocks both the DAG and IP3 limbs of PLC-β signaling stimulated by multiple Gq-coupled receptors by ≥50% (Figure 3A and Figures S1D–F and S3). Acute stimulation of WNK1 activity by IGF1 potentiates PLC-β signaling by ~2-fold (Figures 7A and 7B). WNK1 thus controls a major mode of desensitization of Gq signaling in addition to modulating acute responses. PIP2 itself is a signaling molecule that regulates many plasma membrane targets . Increasing PIP2 synthesis by WNK1 is thus an important mechanism for increasing cellular DAG and IP3 concentrations without causing untoward effects of decreases in membrane PIP2 on other targets.
PLC-β enzymes function as co-incidence detectors that synergistically integrate multiple upstream inputs including Gαq, Gβγ, Ca2+, and Rac [35, 38, 39]. These mechanisms of synergism are results of combined super-additive stimulation of the catalytic activity of PLC-β. In contrast, synergism between WNK1 and Gαq depends on the ability of WNK1 to enhance substrate supply for PLC-β. This system is analogous to the interaction between PLCs and inositol phosphate kinases in which PLC provides the IP3 starting material that the cell-type-specific kinases then phosphorylate to the appropriate inositol polyphosphates .
The interactive control of PLC-β by WNK and Gq may have important implications in the pathogenesis of hypertension in patients with PHA2 that is caused by mutations of WNK1 that increase WNK1 expression . Gq/11-mediated PLC-β signaling plays a central role in the regulation of vascular tone by virtually all vasoactive hormones , and potentiation of PLC-β signaling by WNK1 would promote vasoconstriction and cause hypertension. The role of WNK1 in the regulation of vascular tone is supported by the report that mice with heterozygous inactivation of Wnk1 display reduced blood pressure without renal Na+ wasting and hypovolemia . Finally, the similarity between the phenotypes of WNK1-knockout and Gαq/Gα11-knockout mice (both lethal at embryonic day ~10.5, with cardiac defects) supports the idea that interaction between the WNK1 and Gαq/11 pathways is important for embryonic development [10, 26].
In conclusion, WNK1 potentiates Gq-PLC-β signaling by stimulating the activity of PI4KIIIα to increase synthesis of PIP2. Phosphorylation of WNK1 by Akt kinase increases its activity in this pathway, and allows tyrosine kinase receptors or other activators of the Akt kinase cascade to regulate G protein-mediated PLC-β signaling. The extent and physiological role of WNK1 in the regulation of PLC-β signaling will probably differ among specialized cells. Our understanding of these interactions should expand as we learn more ways in which WNK1 activity is regulated.
Detailed experimental procedures are in Supplementary Information. Human embryonic kidney (HEK)-293, HeLa, and mouse embryonic fibroblasts were cultured and co-transfected with cDNAs for TRPC6-Flag with or without human M3 muscarinic receptor . Ruptured whole-cell, cell-attached, and inside-out excised patch-clamp recording were performed as described [9, 43]. Intracellular Ca2+ transients were examined using ratiometric fluorescent Ca2+ images of fura-2-loaded cells. PIP2 and IP3 were localized according to fluorescence of a GFP-tagged PLC-δ1-PH domain by confocal microscopy . PLC activity was measured by monitoring hydrolysis of [3H]PIP2 on large unilamellar phospholipid vesicles [22, 35]. PI4KIIIα activity from HeLa cells was measured by phosphorylation of phosphatidylinositol (PI) in PI/Triton X-100-mixed micelles .
This study was supported by the National Institutes of Health (GM30355 to E.M.R. and DK59530, DK85726 to C.L.H), the Welch Foundation (I-0982 to E.M.R.) and Genzyme, Inc. (GRIP grant to C.L.H.). We are particularly indebted to Barbara Barylko and Joseph Albanesi for purification of PI4KIIIβ and for continuing help and advice. We thank Melanie Cobb for anti-WNK1 antibody, Tamas Balla for GFP-PLC-δ1-PH, Melvin Simon for Gαq/11-null mouse fibroblasts, and Anna Dugas for technical assistance.
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